Heptaose compounds and preparation thereof

ABSTRACT

The present invention provides compounds of the general formula: ##STR1## in which R and R 1 , independently of one another, each represent a straight-chained or branched alkyl or alkoyl radical containing up to 6 carbon atoms or a phenyl radical or R and R 1  together also form a methylene bridge, the hydrogen atoms of which, independently of one another, can each be substituted by an alkyl radical containing up to 5 carbon atoms or a phenyl radical, R 2  represents an oligoglucoside residue containing 2 to 7 glucose units and X is a hydrogen atom or an optically-determinable residue. 
     The present invention also provides a process for the preparation of these compounds.

The present invention is concerned with new heptaose compounds and withthe preparation thereof, as well as with the use thereof as substratesfor the determination of α-amylase.

The determination of the α-amylase level in serum is an importantclinical parameter for the pancreas function. The commercially-availablereagents for the determination of α-amylase were previouslypreponderantly based on the use of starch or starch derivatives assubstrates. However, these substrates have proved to be unsatisfactory,especially with regard to their uniformity. In order to overcome thisdisadvantage, starch and starch derivatives have been replaced byoligosaccharides and optically-determinable derivatives thereof,maltotetraose, -pentaose, -hexaose and -heptaose and derivatives thereofhaving, in particular, provided interesting improvements (see publishedFederal Republic of Germany Patent Application Nos. 27 41 192 and 27 55803 and U.S. Pat. Nos. 3,879,263 and 4,000,042).

An especially interesting embodiment of the α-amylase determination withthe use of the said oligoglucosides is provided in the presence ofα-glucosidase since a complete breakdown of the oligoglucoside toglucose can hereby be achieved and the glucose can then be easilydetermined by means of processes known for this purpose (cf. publishedFederal Republic of Germany Patent Application No. 27 41 192).

However, it has been found that the adjuvant enzyme α-glucosidasereduces the storage life of the finished reagent mixture since, evenwithout the action of the α-amylase, it brings about a certain splittingof the oligoglucoside.

Therefore, it is an object of the present invention to overcome thisdisadvantage and to provide an α-amylase substrate which is alsosufficiently storage-stable in the presence of α-glucosidase andimproves the correctness of the α-amylase detection.

Thus, according to the present invention, there are provided compoundsof the general formula: ##STR2## in which R and R₁, independently of oneanother, each represent a straight-chained or branched alkyl or alkoylradical containing up to 6 carbon atoms or a phenyl radical or R and R₁together also form a methylene bridge, the hydrogen atoms of which,independently of one another, can each be substituted by an alkylradical containing up to 5 carbon atoms or a phenyl radical, R₂represents an oligoglucoside residue containing 2 to 7 glucose units andX is a hydrogen atom or an optically-determinable residue, especially anitrophenyl radical.

We have found that, with the compounds according to the presentinvention, the reagent ready for use, even in the presence ofα-glucosidase, does not undergo any changes and, therefore, even after along time, provides correct α-amylase values.

Examples of alkyl radicals in the compounds (I) include methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, its isomers,n-hexyl, its isomers, as well as the cyclohexyl radical. In the sameway, when the substituents on the oxygen atoms in the 4- and/or6-positions of the terminal glucoside residue are alkoyl radicals, thesepreferably correspond to the above alkyl radicals. Preferred compoundsaccording to the present invention are those in which R and R₁ togetherform an optionally substituted methylene bridge and especially preferredare those which are substituted by an alkyl or phenyl radical, anethylidene or benzylidene radical being particularly preferred.

Of the oligoglucoside residues R₂, those are preferred which contain 3,4 and 6 glucose units.

If X is an optically-determinable residue, it can be a residue whichitself displays a colour in the visible or in the UV range or a residuewhich becomes optically determinable by reaction with a furthercompound, for example by conversion into a coloured material or bycoupling with a coloured material. Such optically-determinable residuesare known and do not here require any further explanation. Nitrogroup-containing phenyl radicals, such as nitrophenyl or3,4-dinitrophenyl radicals, are preferred.

The preparation of the compounds according to the present invention cantake place starting from oligoglucosides containing 3 to 8 glucose unitswhich terminally optionally carry an optically-determinable group X,wherein these are reacted under esterification or etherificationconditions with an orthooxy compound, preferably with a dialkoxyethaneor a corresponding benzyl derivative, the formation of a compound ofgeneral formula (I), in which R and R₁ together form an optionallysubstituted methylene radical and possibly subsequently free hydroxylgroups are blocked, for example by peracetylation, the methylene bridgeis split at the oxygen atom in 4- or 6-position and, if necessary, thefree hydroxyl group thus formed in the 4- or 6-position is againetherified or esterified or the product is transetherified ortransesterified and other hydroxyl-blocking groups, for example acetylradicals, are subsequently split off.

The compounds with an optionally substituted methylene bridge arising asintermediate products in the case of this synthesis but otherwise alsopreferred as end products can, according to the present invention, beprepared by reacting a compound of the general formula:

    R.sub.3 -X                                                 (II)

in which X has the above-given meaning and R₃ is an oligoglucosideresidue containing 3 to 8 glucose units, in the presence ofp-toluenesulphonic acid with a compound of the general formula: ##STR3##in which R₄ and R₅, independently of one another, each signifies ahydrogen atom or an alkyl radical containing up to 5 carbon atoms or aphenyl radical, in a polar organic solvent.

The polar organic solvent used is preferably dimethylformamide orformamide but other polar organic solvents with a comparable basicitycan also be used.

Surprisingly, the reaction gives rise to uniform products without thenumerous hydroxyl groups present having to be protected. Possibly formedby-products can be separated off without difficulty.

The reaction is preferably carried out at a temperature of from about10° to about 70° C., ambient temperature being especially preferred.Since by-products are scarcely formed by the reaction, for theachievement of the best yields, it is preferable to use a stoichiometricexcess of the compound of general formula (III).

Examples of compounds of general formula (III) include dimethoxymethane,dimethoxyethane, diethoxyethane, dipropoxyethane, dibutoxyethane,dimethoxypropane, dimethoxyisopropane and phenyldimethoxymethane.

Examples of compounds of general formula (II) include maltotetraose,maltopentaose, maltohexaose, maltoheptaose and the derivatives thereofterminally substituted by an optically-determinable group, such asmononitrophenylmaltoheptaose, 3,4-dinitrophenylmaltoheptaose and thelike.

The superior storage stability of the compounds according to the presentinvention is shown by the fact that under the conditions of a widelyused, commercially available α-amylase colour test withp-nitrophenylmaltoheptaoside as substrate (G₇ pNP) namely: buffer pH7.1; sodium chloride 50 mM; about 30 U/ml. α-glucosidase; and 5 mMsubstrate, in the case of practically identical courses of reaction withregard to lag phase and linearity, with4,6-ethylidene-p-nitrophenylmaltoheptaoside (Eth-G₇ pNP), within thecourse of 4 days as 25° C., practically no glucose is formed, whereaswith G₇ pNP, a noticeable splitting to glucose takes place. Under thesame conditions at 4° C., with the substrate according to the presentinvention, after 8 days there is no glucose formation but in the case ofthe comparison substrate, a noticeable splitting occurs. Therefore,according to the present invention, new compounds are provided which, assubstrates for α-amylase in the presence of α-glucosidase, displaysuperior properties.

The following Examples are given for the purpose of illustrating thepresent invention:

EXAMPLE 1 Process for the preparation of4,6-ethylidene-4-nitrophenyl-α-D-maltoheptaoside Batch

250 g. (196 mMol) 4-nitrophenyl-α-D-maltoheptaoside

31.5 ml. (297 mMol) acetaldehyde dimethylacetal

20 g. p-toluenesulphonic acid monohydrate

1.5 liters dimethylformamide (DMF)

Synthesis

250 g. 4-nitrophenyl-α-D-maltoheptaoside and 20 g. p-toluenesulphonicacid monohydrate are dissolved in about 1.5 liters DMF and, forrendering anhydrous, are evaporated to dryness on a rotary evaporator,the water content thereby decreasing from 0.4% to 0.02%.

The residue is dissolved in 1.5 liters DMF, 31.5 ml. acetaldehydedimethyl acetal are added thereto and the reaction mixture is firststirred at 50° C. for 9 hours and then maintained for about 10 hours atambient temperature. The reaction mixture is evaporated to dryness andthe residue is dissolved in water, adjusted with lithium hydroxidesolution to pH 7.3, clarified by filtration and evaporated to 750 ml.

The HPLC analysis of the product shows a content of about 75 to 80% ofethylidene-4-nitrophenyl-α-D-maltoheptaoside with about 20% of startingmaterial.

Chromatographic purification

The sample solution (750 ml.) is applied to a 150×25 cm. column with 70liters "Dowex" 50 WX2 (200 to 400 mesh) lithium⁺ and eluted with waterat a flow rate of 1.5 liters/hour. 4.5 liter fractions are therebycollected, the chromatography being monitored by means of a UV detectorat 280 nm.

After about 100 liters, unreacted 4-nitrophenyl-α-D-maltoheptaoside iseluted and after about 200 liters the desired product is eluted.

The fractions which contain the desired product are combined andevaporated to dryness. The residue is dissolved in 1.5 liters methanol,filtered and precipitated at 0° C. with 4 liters isopropanol and 10liters petroleum ether. After stirring overnight at 4° C., the productis filtered off with suction, washed with isopropanol and petroleumether and dried at 30° C. in a drying cabinet.

Yield: 150 g. (58% of theory) of colourless powder.

Molecular weight 1300.

Water content (according to K. Fischer) 4.5%.

Isopropanol (gas chromatographically) 5%

Acetaldehyde after acidic hydrolysis (enzym.) 91%

[α]_(D) ²⁵ referred to dry substance=77° (c=1, H₂ O)

HPLC: 99 surface percent (5μ-NH₂ column; acetonitrile/water 1:1 v/v, 1mMol/1 o-phosphoric acid, detection at 305 nm).

EXAMPLE 2 Process for the preparation of 4,6-ethylidenemaltoheptaoseBatch

10 g. (8.7 mMol) maltoheptaose

1.8 ml. (17 mMol) acetaldehyde dimethyl acetal

1 g. p-toluenesulphonic acid monohydrate

75 ml. DMF

Synthesis

10 g. Maltoheptaose and 1 g. p-toluenesulphonic acid monohydrate aredissolved in 100 ml. DMF and evaporated to dryness. In this way, theresidue is freed from water. The residue is dissolved in 75 ml. DMF,mixed with 1.7 ml. acetaldehyde dimethyl acetal and stirred for 15 hoursin a closed vessel at 50° C. The reaction solution is then evaporated todryness and the residue is dissolved in water, neutralised with lithiumhydroxide to pH 7, filtered clear and concentrated to 50 ml.

Chromatographic purification

The 50 ml. sample solutions are applied to a chromatography column(180×5 cm.) with 3.5 liters "Dowex" 50 WX2 (200 to 400 mesh) Li⁺ andeluted with water at a flow rate of 150 ml./hour. 30 ml. fractions arecollected which run through a UV detector (280 nm).

After 1.8 liters, maltoheptaose is eluted and after 2.25 litersethylidene-maltoheptaose is eluted.

The main fractions are combined and evaporated to dryness in a rotaryevaporator. The residue is dissolved in 200 ml. methanol with theaddition of some water, mixed with 200 ml. isopropanol and stirred at 4°C., the product thereby precipitating out. It is filtered off withsuction, washed with isopropanol and petroleum ether and dried in avacuum at 25° C. over phosphorus pentoxide.

Yield: 6.2 g. (60% of theory).

Analysis: water content (according to K. Fischer) 7.6%.

Acetaldehyde after acidic hydrolysis (enzym.) 91.2%.

[α]_(D) ²⁵ referred to dry substance (c=1, H₂ O)=69°.

HPLC (conditions see Example 1): 96 surface percent.

The reaction with tetrazolium blue demonstrates that the reducing end isfree.

EXAMPLE 3

Reagent: 682.5 mg. ethylidene-G₇ PNP (5.25 mMol/liter) are dissolved in100 ml. sodium phosphate buffer (105 mMol/liter), containing sodiumchloride (52.5 mMol/liter), as well as α-glucosidase (42 U/ml.), andadjusted to pH 7.10.

End concentration in the test: phosphate buffer 100 mMol/liter, sodiumchloride 50 mMol/liter, substrate 5 mMol/liter, α-glucosidase 40 U/ml.Test batch: To 2.0 ml. of reagent tempered to 25° C. are added 0.1 ml.of sample and the mixture is tempered to 25° C. After a pre-incubationtime of 4 minutes (lag phase), the extinction increase at Hg 405 nm isregistered with a recoder on an Eppendorf photometer. From theextinction change per minute (ΔE/min.) is calculated the activity of theamylase in the samples according to the following formula: ##EQU1##

Recovery again of α-amylase: The determination of the activity isrepeated with reagent stored at 4° C. and at 25° C. for definiteintervals of time, the following values being found:

    ______________________________________                                                     human     human     control serum                                storage of the                                                                             serum 1   serum 2   PNU 572                                      reagent      U/l.      U/l.      U/l.                                         ______________________________________                                        starting value                                                                             118       383       214                                          4 hours at 25° C.                                                                   120       379       214                                          8 hours at 25° C.                                                                   120       391       216                                          24 hours at 25° C.                                                                  113       360       209                                          32 hours at 25° C.                                                                  120       392       217                                          48 hours at 25° C.                                                                  126       382       214                                          8 hours at 4° C.                                                                    119       370       210                                          24 hours at 4° C.                                                                   118       387       215                                          48 hours at 4° C.                                                                   123       393       208                                          72 hours at 4° C.                                                                   113       401       223                                          80 hours at 4° C.                                                                   121       375       219                                          average value/VK                                                                           119 3.2%  383 3.0%  214 2.0%                                     ______________________________________                                    

The individual values lie within the usual variation breadth for manualdeterminations of enzyme activities.

We claim:
 1. A compound of the formula: ##STR4## in which R and R₁,independently of one another, each represent a straight-chained orbranched alkyl or alkoyl radical containing from 1 to 6 carbon atoms ora phenyl radical or R and R₁ together form a methylene bridge, thehydrogen atoms of which, independently of one another, can each besubstituted by an alkyl radical containing up to 5 carbon atoms or aphenyl radical, R₂ represents an oligoglucoside residue containing 2 to7 glucose units and X is a hydrogen or a nitrophenyl residue.
 2. Thecompound of claim 1 wherein X is nitrophenyl.
 3. The compound of claim 1wherein X is 3,4-dinitrophenyl.
 4. The compound of claim 1 wherein X ishydrogen.
 5. The compound of claim 1, wherein at least one of R and R₁is an alkyl radical of from 1 to 6 carbon atoms.
 6. The compound ofclaim 5, wherein said alkyl radical is selected from the groupconsisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,t-butyl, n-pentyl and its isomers, n-hexyl and its isomers andcyclohexyl.
 7. The compound of claim 1, wherein at least one of R and R₁is an alkoyl radical of from 1 to 6 carbon atoms.
 8. The compound ofclaim 7, wherein said alkoyl radical is a derivative of an alkylselected from the group consisting of methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl and its isomers, n-hexyland its isomers and cyclohexyl.
 9. The compound of claim 1, wherein atleast one of R and R₁ is a phenyl radical.
 10. The compound of claim 1,wherein R and R₁ form a methylene bridge.
 11. The compound of claim 10,wherein said methylene bridge is substituted by an alkyl radical of from1 to 5 carbon atoms or a phenyl radical.
 12. The compound of claim 1,wherein R₂ contains 3, 4 or 6 glucose residues.
 13. The compound ofclaim 1, wherein said compound is4,6-ethylidene-4-nitrophenyl-α-D-maltoheptaoside.
 14. The compound ofclaim 1, wherein said compound is 4,6-ethylidene maltoheptaoside. 15.The compound of claim 11, wherein R and R₁ together form a methylenebridge substituted by a phenyl radical to form a benzylidene.
 16. Thecompound of claim 11, wherein R and R₁ together form a methylene bridgesubstituted by a methyl radical to form an ethylidene.
 17. A process forthe preparation of the compound of claim 1, comprising the stepsofreacting a compound of the formula:

    R.sub.3 -X                                                 (II)

in which X has the meaning given in claim 1 and R₃ is an oligoglucosideresidue containing 3 to 8 glucose units, in the presence ofp-toluene-sulphonic acid, with a compound of the formula: ##STR5## inwhich R₄ and R₅, independently of one another, each signifies a hydrogenatom or an alkyl radical containing up to 5 carbon atoms or a phenylradical, in a polar organic solvent.
 18. The process of claim 17,wherein the organic solvent is dimethylformamide.
 19. The process ofclaim 18, wherein the reaction is carried out at a temperature of 10° to70° C.
 20. The process of claim 17, wherein an excess stoichiometricamount of the compound of formula (III) is present during the reaction.21. The process of claim 17 wherein the reaction is carried out at atemperature of 10° to 70° C.